dodecane-12-lactam, manufacturing of, by-products from, distillation residues

Endpoint summary

Description of key information

There is a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test performed according to OECD 422 guideline and  GLP (Spézia, 2012). The males and females were treated at 100, 300 and 1000 mg/kg/day for at least 5 to 8 weeks.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

December 2011-July 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This study has been performed according to OECD and/or EC guidelines and according to GLP principles.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was administered as a suspension in the vehicle. The test item was ground to a fine powder, using a mortar and pestle, and then mixed with the required quantity of vehicle.
The test item dosage forms were prepared daily in the vehicle.
The dosage forms were delivered to the study room at room temperature.

VEHICLE
- Concentration in vehicle: 20, 60 and 200 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg/day.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Type of method: HPLC-UV,
The concentration of the lactame content in each control and test item dosage form prepared for use in weeks 1, 3 and 5 were determined (three occasions). Concentration of test item in the dosage forms were then determined accordingly.
The dosage form preparations were delivered to the study room before acceptance of the results of the concentration.

Homogeneity: the dosage forms containing the test item in 0.5% methylcellulose in drinking water treated by reversed osmosis at 5 and 200 mg/mL were found to be homogeneous.
Stability: not assessed, dose formulation prepared daily

Duration of treatment / exposure:

The dosage forms were administered daily according to the following schedule:
in the males:
- 2 weeks before pairing,
- during the pairing period (3 weeks),
- until sacrifice (at least 5 weeks in total),

in the females:
- 2 weeks before pairing,
- during the pairing period (3 weeks),
- during gestation,
- during lactation until day 5 p.p. inclusive,
- until sacrifice for females with no delivery.

Day 1 corresponds to the first day of the treatment period.

Frequency of treatment:

daily

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

10 animals per sex per group.

Details on study design:

Three groups of ten male and ten female Sprague-Dawley rats received the test item, Dodecane-12-Lactam Manufacturing of, by-products from, distillation residues, daily, by oral (gavage) administration, before mating, during mating and, for the females, throughout gestation until day 5 p.p., at dose-levels of 100, 300 or 1000 mg/kg/day. An additional group of ten males and ten females received the vehicle control, 0.5% methylcellulose aqueous solution, under the same experimental conditions. The dosing volume was 5 mL/kg/day.

Positive control:

not appropriate.

Observations and examinations performed and frequency:

Animals were checked daily for clinical signs, mortality, and detailed clinical observations were conducted weekly. Body weights and food consumption were recorded weekly until mating and then at designated intervals throughout gestation and lactation.
The animals were paired for mating after 2 weeks of treatment and the dams were allowed to litter and rear their progeny until day 5 p.p.
FUNCTIONAL OBSERVATION BATTERY: The first five males and the first five females were evaluated once at the end of the treatement period. The FOB included touch response, forelimb grip strength, pupillary reflex, visual stimulus response, auditory startle reflex, tail pinch response, righting reflex, landing foot splay, rectal temperature and motor activity.
LABORATORY INVESTIGATIONS: Prior to sacrifice, blood samples were also taken from these animals for analysis of hematology, urinalysis and blood biochemistry parameters.

Sacrifice and pathology:

The males were sacrificed after completion of the mating period. Body weights and selected organs weights were recorded and a complete macroscopic post-mortem examination performed, with particular attention paid to the reproductive organs. A microscopic examination was also conducted on selected organs from the first five males in the control group and the high-dose group. Microscopic examination was conducted on all macroscopic lesions from all groups.
Based upon the microscopic results of the high-dose group, liver and spleen tissues of the first five animals of the low- and intermediate-dose groups were examined.

Dams were sacrificed on day 6 p.p.. Body weights and selected organs weights were recorded and a complete macroscopic examination was performed, with particular attention paid to the reproductive organs.

MICROSCOPIC EXAMINATION:
A microscopic examination was performed on:

. all tissues listed in the tissue procedure table from the first five males sacrificed and the first five females that delivered and were sacrificed on day 6 p.p. of the control and high-dose
groups (groups 1 and 4),
. all tissues listed in the tissue procedure table from the female rat (X21461) given 100 mg/kg/day sacrificed because of difficulty to deliver and the female rat (X21478) given 300 mg/kg/day sacrificed because of no delivery, to investigate possible causes,
. all macroscopic lesions of all the animals of the low- and intermediate-dose groups (groups 2 and 3) sacrificed on completion of the treatment period,
. kidneys (males), liver (both sexes) and mesenteric lymph nodes (both sexes) of the first five males sacrificed and the first five females that delivered and were sacrificed on day 6 p.p. of the low- and intermediate-dose groups.

Special emphasis was paid to the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure.

Statistics:

Citox software (version D.05) was used to performed statistical analyses of hematology, blood biochemistry and urinalysis data with the Kolmogorov-Lilliefors test, Bartlett and Dunnett.

The other data were compared by one-way analyisis of variances and Dunnett test (mean values being considered as normally distributed, variances being considered as homogenous) or by Fisher exact probability test (proportions).

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

excessive ptyalism

Mortality:

mortality observed, treatment-related

Description (incidence):

excessive ptyalism

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

non adverse

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

non adverse

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

non adverse

Histopathological findings: neoplastic:

no effects observed

Details on results:

Results

The analytical procedure for determination of the Dodecane-12-Lactam Manufacturing of, by-products from, distillation residues is based on the determination of the lactame content in the analytical samples.

The lactame concentrations in the administered dosage forms analyzed in weeks 1, 3 and 5 remained within an acceptable range of -6.9% to +0.1% when compared to the nominal values (± 15%).
Lactame was not detected in control samples. Mortality
There were no treatment-related deaths.

Clinical signs
Overall, excessive salivation (ptyalism) was observed in males treated from 300 mg/kg/day and in
females at 1000 mg/kg/day. While treatment-related, ptyalism was considered to be non-adverse at
300 mg/kg/day taking into account the incidence and duration of this finding at this dose-level.

Body weight and food consumption
There were no treatment-related effects on mean body weight and mean body weight change neither in males nor in females.

Functional Observation Battery and motor activity
There were no treatment-related effects.

Hematology
There were no significant toxicological effects on mean hematology parameters.

Blood biochemistry and urinalysis
There were no significant toxicological effects on mean blood chemistry parameters and urinalysis

Pairing, mating and fertility
There were no treatment-related effects on pairing, mating and fertility parameters.

Pup observations
There were no effects on live birth, viability and lactation indexes. There were no treatment
related findings in pups at necropsy.

Pathology
There were no treatment-related macroscopic findings.
. Higher relative liver weights were noted in females treated at 1000 mg/kg/day. This weight difference correlated with hepatocellular hypertrophy and was considered to be treatment-related. Minimal to slight hepatocellular hypertrophy was noted in the liver in five out of five males examined treated at 1000 mg/kg/day and in females from 100 mg/kg/day onwards. As this finding was not associated with any hepatocellular degeneration/necrosis, this hypertrophy was considered not to be adverse.
. Macrophages aggregates were observed in the mesenteric lymph nodes, most of the time with lymphoid hyperplasia (increased size/numbers of germinal centers and expansion of the paracortex area) from 100 mg/kg/day in males and from 300 mg/kg/day in females.
These findings suggested inflammation and reactive lymphoid hyperplasia, possibly as a result of test item draining from the digestive tract.
. Hyaline tubular droplets were seen in the kidneys in one out of six males treated at 300 mg/kg/day
and in the six males examined that were treated at 1000 mg/kg/day. As there were no biochemical or hematological changes, this finding was considered not to be adverse.
These microscopic findings were not considered as adverse effects.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: The findings observed in the liver, kidneys and mesenteric lymph node were considered as non adverse

Key result

Critical effects observed:

no

Lowest effective dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Treatment related:

no

Motor activity :

 

Sex                                                              Male                                              Female

Dose-level(mg/kg/day)             0        100        300      1000        0         100         300       1000

 

Number of animals Horizontal movements Mean	5     496	5     490	5     732	5     567	5     468	5     423	5     405	5     451
SD	82.1	202.4	175.7	202.9	190.0	203.7	173.3	212.4
Rearing Mean	158	122	168	131	127	104	137	87
SD	43.3	29.8	39.9	20.6	27.2	22.2	44.5	62.3

 

Hematology

 

Sex                                                          Male                                                   Female

 

Dose-level (mg/kg/day)	0	100	300	1000	0	100	300	1000
Pack cell volume (L/L)	0.45	0.47*	0.47	0.47*	0.42	0.42	0.41	0.42
Thrombocytes (G/L)	958	1060	1147*	1038	1319	1227	1384	1384

Statistically significant:*: p<0.05.

 

 

Overall, there were no dose-related differences in hematological parameters when compared with control group. Therefore a test item treatment-related effect was considered unlikely.

 

 

 Blood biochemistry

 

Sex Dose-level (mg/kg/day)	0	100	Male 300	1000	0	Female 100	300	1000
Glucose (mmol/L)	6.81	6.42	6.23	5.69*	5.90	6.42	5.98	6.03
Bileacid (µmol/L)	9.4	8.7	9.3	6.6*	17.9	23.7	18.5	14.5
ALP (IU/L)	227	265	295*	232	134	152	136	130

ALP:Alkalinephosphatase,Statistically significant: *:p<0.05.

 

 

Conclusions:

The test item, Dodecane-12-Lactam Manufacturing of, by-products from, distillation residues, was administered daily by oral gavage to male and female Sprague-Dawley rats, for 2 weeks before mating, during mating, and until sacrifice (for males) or throughout gestation and until day 5 post-partum (for females), at dose-levels of 100, 300 or 1000 mg/kg/day.
The test item, Dodecane-12-Lactam Manufacturing of, by-products from, distillation residues, was administered daily by oral gavage to male and female Sprague-Dawley rats, for 2 weeks before mating, during mating, and (for females) throughout gestation and until day 5 post-partum, at dose-levels of 100, 300 or 1000 mg/kg/day.

In parent animals, there were effects in the liver, mesenteric lymph nodes and in the kidneys.
. in pups, there were no treatment-related findings.

Based on the experimental conditions of this study:
. the No Observed Adverse Effect Level (NOAEL) for parental toxicity was considered to be
1000 mg/kg/day,
. the No Observed Effect Level (NOEL) for reproductive performance (mating and fertility)
was considered to be 1000 mg/kg/day in the absence of significant effects on mating and fertility at this dose-level,
. the No Observed Effect Level (NOEL) for toxic effects on progeny was considered to be
1000 mg/kg/day in the absence of any treatment-related effect on pups at this dose-level



The No Observed Adverse Effect Level (NOAEL) for parental toxicity was considered to be 1000 mg/kg/day,

Executive summary:

The test item, Dodecane-12-Lactam Manufacturing of, by-products from, distillation residues, was administered daily by oral gavage to male and female Sprague-Dawley rats, for 2 weeks before mating, during mating, and (for females) throughout gestation and until day 5 post-partum, at dose-levels of 100, 300 or 1000 mg/kg/day.

There were no treatement related changes on body weight, food consmuption, motor activity, neurobehaviour, hematology or biochemistry endpoints.

In parent animals, the changes in the liver, mesenteric lymph nodes and in the kidneys were not considered as adverse effects. Based on the experimental conditions of this study:

The No Observed Adverse Effect Level (NOAEL) for parental toxicity was considered to be 1000 mg/kg/day, .

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Additional information

The test item, Dodecane-12 -Lactam Manufacturing of, by-products from, distillation residues, was administered daily by oral gavage to male and female Sprague-Dawley rats, for 2 weeks before mating, during mating, and for (females) throughout gestation and until day 5 post-partum at dose-levels of 100, 300 or 1000 mg/kg/day. The exposure duration for males and females were at least 5 to 8 weeks dosing. The animals were subjected to detailed clinical examination including functional observation battery and motor activity. Blood sampling was also performed for hematology and biochemistry analyses at the end of dosing on the day of sacrifice. A complete macroscopic and microscopic examination was performed on listed tissues.Overall, excessive salivation (ptyalism) was observed in males treated from 300 mg/kg/day and in females at 1000 mg/kg/day. There were no effects on mean body weight and food consumption and no effects on functional observation battery and motor activity. No changes were observed on blood biochemistry , hematology and urinalysis parameters. The variations observed in the liver (minimal to slight hyperthrophy), in the kidneys (hyaline droplets in males at the high-dose levels) and in the mesenteric lymph nodes were not considered as adverse effects. Therefore the No Observed Adverse Effect Level (NOAEL) for parental toxicity was considered to be 1000 mg/kg/day.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
The males and females were dosed during at least 5 to 8 weeks in this OECD 422 guideline study. Motor activity, Hematology and biochemistry investigations toguether with microscopic examinations were performed. The changes observed were not considered as adverse effects